Preparation of Mouse Embryos for Optical Projection Tomography Imaging

INTRODUCTION

Optical projection tomography (OPT) is a method for three-dimensional (3D) imaging of whole vertebrate embryos. The specimen is suspended in an index-matching liquid to reduce the scattering of light and reduce heterogeneities of refractive index throughout the specimen. Inside the OPT scanning device, the specimen is maintained within the liquid and rotated through a series of angular positions, and an image is captured at each orientation. There are two imaging modes for OPT: transmission imaging or bright-field OPT, and emission tomography or fluorescence OPT. In both modes of OPT, the fact that lenses are used to focus an image means that OPT suffers one technical drawback compared to its X-ray equivalent: As with all optical imaging systems, there is only a limited depth of focus, and this usually cannot encompass the entire specimen. OPT, therefore, generally takes advantage of a compromise, which yields good results—the focal plane is positioned halfway between the axis of rotation and the edge of the specimen closest to the lenses. This maximizes the focused information obtained from the specimen, while bypassing the need to image at multiple depths and keeping the imaging time to a minimum. This article presents a series of short methods for preparing specimens for OPT imaging. These include fixing the sample, embedding it correctly in an agarose block, trimming the agarose block, and, finally, clearing for optical imaging.